AbstractResistance of carcinoma cells to anoikis, apoptosis that is normally induced by detachment of nonmalignant epithelial cells from the extracellular matrix, is thought to be critical for carcinoma progression. Molecular mechanisms that control anoikis of nonmalignant and cancer cells are understood poorly. In an effort to understand them we found that detachment of nonmalignant intestinal epithelial cells triggers upregulation of Chk2, a pro‐apoptotic protein kinase that has never been implicated in anoikis and has been thought to kill cells mainly under the conditions compromising genome integrity. We found that enforced downregulation of Chk2 protects intestinal epithelial cells from anoikis. Chk2 can kill cells by stabilizing p53 tumor suppressor protein or via p53‐independent mechanisms, and we established that Chk2‐mediated anoikis of intestinal epithelial cells is p53‐independent. We further found that, unlike nonmalignant intestinal epithelial cells whose anoikis is triggered by detachment‐induced Chk2 upregulation, intestinal epithelial cells carrying oncogenic ras, a known inhibitor of anoikis, remain anoikis‐resistant in response to enforced Chk2 upregulation. By contrast, drugs, such as topoisomerase I inhibitors, that can kill cells via Chk2‐indpendent mechanisms, efficiently triggered anoikis of ras‐transformed cells. Thus, oncogenic ras can prevent Chk2 from triggering anoikis even when levels of this protein kinase are elevated in cancer cells, and the use of therapeutic agents that kill cells in a Chk‐2‐independent, rather than Chk‐2‐dependent, manner could represent an efficient strategy for overcoming ras‐induced anoikis resistance of these cells. We conclude that Chk‐2 is an important novel component of anoikis‐promoting machinery of intestinal epithelial cells.